Interleukin-10 (IL-10) has potent immunoregulatory effects on the maturation and the antigen-presenting cell (APC) function of dendritic cells (DCs). The molecular basis underlying these effects in DCs, however, is ill defined. It is well established that the transcription factor NF-kappaB is a key regulator of DC development, maturation, and APC function. This study was initiated to determine the effects of IL-10 on the NF-kappaB signaling pathway in immature DCs. IL-10 pretreatment of myeloid DCs cultured from bone marrow resulted in reduced DNA binding and nuclear translocation of NF-kappaB after anti-CD40 antibody or lipopolysaccharide (LPS) stimulation. Furthermore, inhibited NF-kappaB activation was characterized by reduced degradation, phosphorylation, or both of IkappaBalpha and IkappaBepsilon but not IkappaBbeta and by reduced phosphorylation of Ser536, located in the trans-activation domain of p65. Notably, IL-10-mediated inhibition of NF-kappaB coincided with suppressed IkappaB kinase (IKK) activity in vitro. Furthermore, IL-10 blocked inducible Akt phosphorylation, and inhibitors of phosphatidylinositol 3-kinase (PI3K) effectively suppressed the activation of Akt, IKK, and NF-kappaB. These findings demonstrate that IL-10 targets IKK activation in immature DCs and that suppressing the PI3K pathway in part mediates blockade of the pathway.